Developmental decisions are controlled by the combined action of multiple regulatory pathways. One critical aspect of regulation is the controlled timing of the onset of specific developmental programs that render cells competent for responding to other signals. Our goal is to understand the genetic and biochemical mechanisms by which several genes regulate developmental timing and to understand the link between the regulatory pathway and specific cellular events such as cell divisions and differentiations. Research under this grant has used C. elegans vulval induction as a model system to investigate the functions and interactions of multiple pathways, including the Ras-MAP kinase pathway. Recently, by searching for genes acting downstream of Ras signaling, we have identified four genes that are involved in regulating the timing of vulval cell divisions. Among them, LIN-66 represses the expression of a key timing regulator LIN-28, and AIN-1 binds to ALG-1, Dicer and microRNA of the microRNA induced complexes (miRICS) and targets ALG-1 to specific cytoplasmic foci. A transcription factor LIN-31 that acts downstream of Ras-MAPkinase is also involved in timing regulation. We will carry out a series molecular and genetic experiments to understand how LIN-66 regulates lin-28 expression and what biochemical properties AIN-1 provides to miRISCs. Through identifying the miRNAs and their targets associated with AIN-1, we will determine if AIN-1 is associated with only a subset of miRNAs for their functions, as well as learning about these specific functions carried by these targets. We also plan to study the functions of AIN-2, which has structural and functional similarities to AIN-1, as well as two other genes. Finally, we will search for targets of LIN-31 to learn about what factors execute the regulatory role of LIN-31 in timing regulatory and in mediated Ras signaling. microRNA functions and Ras signaling are conserved between worms and human, and are involved in many human developmental processes and diseases, such as cancers. Studies in C. elegans have made a huge impact on the research in related fields. The proposed research intends to provide new insights into cellular processes involved in these newly identified genes.